(i) Field of the Invention
The present invention relates to an improved method for purifying L-phenylalanine which is suitable for the purification of L-phenylalanine produced by a reaction using cinnamic acid as a starting material and phenylalanine ammonia lyase in the presence of ammonia.
(ii) Description of the Prior Art
L-phenylalanine is an essential amino acid which is used as a medicine such as an amino acid transfusion, and is an important component as a constitutional amino acid of .alpha.-L-aspartyl-L-phenylalanine methyl ester which is a peptide sweetener.
Manufacturing methods of L-phenylalanine can be classified into a chemical synthesis method, a fermentation method and residual an enzyme method. An example of the enzyme method comprises using cinnamic acid as a starting material and phenylalanine ammonia lyase in the presence of ammonia. This reaction is reversible, and cinnamic acid which is the starting material remains in the reaction solution. Therefore, the remaining cinnamic acid must be removed in a purification step to efficiently collect the L-phenylalanine.
As purification methods for L-phenylalanine, there have been employed a method using an ion exchange resin adsorbent (Japanese Patent Application Laid-open No. 194056/1986), a method using concentration/crystallization (Japanese Patent Application Laid-open No. 133893/1985) and a method using a lower alcohol (U.S. Pat. No. 4731469).
The above-mentioned purification methods of L-phenylalanine have the following problems in the case they are utilized on an industrial scale.
In the ion exchange resin adsorption method, the L-phenylalanine is separated from the cinnamic acid by chromatography, as described in Japanese Patent Application Laid-open No. 194056/1986. In this method, however, volumetric efficiency is poor and what is worse, enormous energy is consumed. For these reasons, the method is inconvenient for mass production.
In the above-mentioned method using concentration/crystallization, the pH of an aqueous L-phenylalanine solution containing cinnamic acid is adjusted to 6-9, and after concentration, cooling and crystallization are carried out, as described in Japanese Patent Application Laid-open No. 133893/1985. However, it is difficult to completely separate the cinnamic acid by this method. This Japanese Patent Application Laid-open No. 133893/1985 also describes another method which comprises adjusting the pH of an aqueous L-phenylalanine solution containing cinnamic acid to a level of strong acidity, removing precipitated cinnamic acid, and then purifying L-phenylalanine. However, in order to remove the cinnamic acid, a separating operation such as filtration of the strongly acidic solution must be carried out. Therefore, the operation is troublesome and its volumetric efficiency is low. Since L-phenylalanine high soluble in the strongly acidic state, the solution must to be neutralized to an isoelectric point or its vicinity of L-phenylalanine, after the removal of cinnamic acid. Thus, a large amount of salts is inconveniently formed, even though the cinnamic acid can be removed.
In the method using a lower alcohol, a lower alcohol is added to a concentrated solution of L-phenylalanine, so that the cinnamic acid is dissolved in the lower alcohol and thus it is removed, as described in U.S. Pat. No. 4731469. In this method, however, a large amount of the lower alcohol is necessary, for example, about five times by weight of the L-phenylalanine. Accordingly, the yield of L-phenylalanine lowers, the volumetric efficiency deteriorates, and large facilities for the recovery of the solvent are required, even though cinnamic acid can be removed. In addition, in manufacturing L-phenylalanine, the recovery of unreacted cinnamic acid is a serious problem, but a crystallization filtrate from which L-phenylalanine has been separated contains a large amount of impurities, so that the recovered cinnamic acid inconveniently has a low purity.
Furthermore, the solubility of L-phenylalanine in water is low, and so there remains a problem that when the crystals of L-phenylalanine are deposited from an aqueous L-phenylalanine solution, the volumetric efficiency is very poor.